Micro RNA isolation from a biological sample

ABSTRACT

The present disclosure provides methods and kits for isolating miRNAs from biological fluids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Application Ser. No. 15/038,967filed May 24, 2016, which is a U.S. National Stage Application of PCTInternational Application No. PCT/US2014/067321, filed Nov. 25, 2014,which claims priority to U.S. Provisional Pat. App. Ser. No. 61/985,000,filed Apr. 28, 2014, and U.S. Provisional Pat. App. Ser. No. 61/909,834,filed Nov. 27, 2013, the disclosure of each of which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present disclosure relates to means for isolating microRNAs frombiological fluids.

BACKGROUND

MicroRNAs (miRNAs) are small, noncoding RNAs that influence generegulatory networks by post-transcriptional regulation of specificmessenger RNA (mRNA) targets via specific base-pairing interactions.miRNAs have been shown to be present in human biofluids in a cell-freeform. These cell-free miRNAs may be non-vesicular, bound and protectedby proteins in miRNA-protein complexes, enclosed in membrane-boundvesicles such as exosomes or microvesicles, or both. Given the importantfunctional role of miRNA in disease, this set of nucleic acid moleculescontains candidates for diagnosing and prognosing disease, andmonitoring response to therapies in a wide variety of patients and insubjects prior to manifesting disease in a readily available biologicalsample, such as blood serum and plasma, urine, or saliva. Currentmethods of isolating miRNAs are directed to relatively abundant miRNAsin cells and tissues, use spin columns which are not readily automatedor scaled up, are complicated and involve toxic compounds, or mayspecifically isolate either vesicular or non-vesicular miRNAs.Furthermore, miRNAs of diagnostic or prognostic interest are oftenpresent at low abundance in biofluids, making their detection usingcurrent isolation methods challenging. Therefore, there is a need for asimple, efficient, automatable, and scalable method for isolating all ora majority of miRNAs in biofluids.

SUMMARY OF THE INVENTION

One aspect of the present disclosure provides a method for isolatingmicroRNA (miRNA) from a biological fluid. The method comprisescontacting the biological fluid with a surface active agent and ananti-miRNA-binding protein reagent, wherein the surface active agentdissociates biological fluid components and the anti-miRNA-bindingprotein reagent interacts with a miRNA-binding protein associated withmiRNA to form immunoprecipitated miRNA complexes. The method furthercomprises releasing miRNA from the immunoprecipitated miRNA complexes.

Another aspect of the disclosure encompasses a kit for isolatingmicroRNA from a biological fluid. The kit comprises a surface activeagent, an anti-miRNA-binding protein reagent, and a miRNA releasingreagent.

Other aspects and iterations of the disclosure are described in moredetail below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 presents two plots comparing miRNA isolation from 0.2 ml plasmausing Tri Reagent® BD or RNA immunoprecipitation (RIP). The amounts ofisolated miRNA by RIP are presented as the fold difference from miRNAisolated using Tri Reagent® BD. (A) depicts the levels of let-7a-5p,23a-3p, and 191-5p miRNAs, and (B) shows the levels of 142-3p and 451amiRNAs.

FIG. 2 presents three plots showing levels of miRNAs isolated fromplasma using Ago-RIP or a Qiagen column purification kit (Q1, Q2). Theamounts of isolated miRNA, as copies of miR/μl eluted, are shown forlet-7a, 23a, and 142 miRNAs (A), 191 miRNA (B), and 451a miRNA (C).

FIG. 3 presents three plots showing levels of miRNAs isolated fromplasma using RIP with biotinylated (b-Ago2 or b-Ago) or non-biotinylated(Ago2) antibodies with streptavidin or Protein A beads. Shown are theamounts of isolated miRNA represented as copies of miR/μl eluted forlet-7a (A), 23a (B), and 191 (C) miRNAs. The antibody (clone inparenthesis) used is noted on the x axes.

FIG. 4 presents two plots showing levels of miRNAs isolated from plasmausing Ago-RIP with heat release. Q represents Qiagen columnpurification; RIP-Q represents immunoprecipitation followed by Qiagencolumn purification; bRIP-Q represents immunoprecipitation usingbiotinylated antibody followed by Qiagen column purification. (A)depicts levels of synthetic cel-miR-39-3p spike-in using Qiagen columnpurification, RIP in combination with column purification and RIP withprotease K release. Levels of spike-in are represented as percent totalof synthetic cel-miR-39-3p spiked-in during isolation. (B) depictslevels of let7a miRNA isolated from plasma using Qiagen columnpurification, RIP in combination with column purification and RIP withprotease K release. Levels of let7a are represented as copies of let7ain 1 μl of recovered sample.

FIG. 5 presents two plots showing levels of miRNAs isolated from plasmausing Ago-RIP with protease K release at various temperatures. (A)depicts levels of let7a miRNA released by protease K (left bar at eachtemperature), and levels retained on the beads (right bar at eachtemperature). Levels of let7a miRNA are represented as percent total oflet7a miRNA in 0.2 ml of the same plasma isolated with Qiagen's miRNeasySerum/Plasma Kit. (B) depicts levels of miR451a miRNA released byprotease K (left bar at each temperature), and levels retained on thebeads (right bar at each temperature). Levels of miR451a miRNA arerepresented as percent total of miR451a miRNA in 0.2 ml of the sameplasma isolated with Qiagen's miRNeasy Serum/Plasma Kit.

FIG. 6 presents two plots showing levels of let7a (A) or miR451a (B)miRNA isolated from plasma using commercially available methods of miRNAisolation, or isolation using RIP with protease K release in standardtubes. E1 and E2 represent miRNA isolation using miRCury RNA IsolationKit-biofluids from Exiqon. Q1 and Q2 represent miRNA isolation usingmiRNeasy Serum/Plasma Kit from Qiagen. RIP-std-Q representsimmunoprecipitation followed by Qiagen column purification. Levels ofmiRNA are represented as total copies recovered from 0.2 ml plasma.

FIG. 7 presents two plots showing levels of let7a miRNA isolated fromplasma using commercially available methods of miRNA isolation, orisolation using Ago-RIP with protease K release (RIP1-4). (A) Showstrial 1 and (B) shows trial 2. E1 and E2 represent miRNA isolation usingmiRCury RNA Isolation Kit—Biofluids from Exiqon. Q1 and Q2 representmiRNA isolation using miRNeasy Serum/Plasma Kit from Qiagen. Levels oflet7a are represented as total copies of let7a recovered from 0.2 mlplasma.

FIG. 8 presents two plots showing levels of miRNAs isolated from plasmausing Ago-RIP followed by protease K release with or without proteaseand RNase inhibitors, and with or without detergent pretreatment.+pre,+inh; Igepal and inhibitors added to plasma and incubated ˜30minutes before adding to Ago2-beads. +pre,−inh; Igepal added to plasmawithout inhibitors and incubated ˜30 minutes before adding toAgo2-beads. −pre,+inh; Igepal and inhibitors added to plasma at the sametime as Ago2-beads. −pre,−inh; Igepal added to plasma without inhibitorsat the same time as Ago2-beads. (A) Depicts copies of let7a miRNArecovered, and (B) depicts copies of miR451a miRNA recovered.

FIG. 9 presents a plot showing the levels of free (left bar for eachmiRNA) and vesicular (right bar for each miRNA) for the indicated miRNAsas % total IGEPAL treated miRNAs.

FIG. 10 presents three plots showing levels of miRNAs isolated from 0.2or 0.4 ml plasma using Ago-RIP followed by protease K release (RIP), orfrom 0.2 ml plasma using miRCury RNA Isolation Kit-Biofluids from Exiqon(E). (A) Depicts copies of let7a miRNA recovered, (B) depicts copies ofmiR191 miRNA recovered, and (C) depicts copies of miR451a miRNArecovered.

FIG. 11 presents a plot showing the levels of let7a isolated from plasmausing Ago-RIP followed by protease K release. RIP incubations were atroom temperature for 5, 15, 30, or 60 minutes (5′, 15′, 30′, 60′). Thoseincubated 5, 15, or 30 minutes were all washed 5 times before proteinaseK release. Those incubated 60 minutes were washed 5, 4, 3, 2, or 1 times(5w, 4w, 3w, 2w, 1w). Total yield of let7a recovered from 0.2 ml plasmais shown.

FIG. 12A shows the levels of let7a miRNA isolated from plasma usingAgo-RIP or column-based miRNA isolation kits. Three differentexperiments (Exp) are presented. S1 and S2 represent isolation usingAgo-RIP; E1 and E2 represent isolation using miRCury RNA IsolationKit-Biofluids from Exiqon; and Q1 and Q2 represents isolation usingmiRNeasy Serum/Plasma Kit from Qiagen.

FIG. 12B presents the levels of RNU6 small nuclear RNA and SNORD48 smallnucleolar RNA isolated from plasma using Ago-RIP or column-based miRNAisolation kits. Three different experiments are presented. S1 and S2represent isolation using Ago-RIP; E1 and E2 represent isolation usingmiRCury RNA Isolation Kit-Biofluids from Exiqon; and Q1 and Q2represents isolation using miRNeasy Serum/Plasma Kit from Qiagen.

FIG. 12C shows the levels of GAPDH messenger RNA, RN18S ribosomal RNA,and RN28S ribosomal RNA isolated from plasma using Ago-RIP orcolumn-based miRNA isolation kits. Three different experiments arepresented. S1 and S2 represent isolation using Ago-RIP; E1 and E2represent isolation using miRCury RNA Isolation Kit-Biofluids fromExiqon; and Q1 and Q2 represents isolation using miRNeasy Serum/PlasmaKit from Qiagen.

FIG. 13 presents a plot showing the levels of the indicated miRNAsisolated from plasma via Ago-RIP using anti-Ago1 antibodies, anti-Ago2antibodies, or a combination of both anti-Ago1 and anti-Ago2 antibodies.

DETAILED DESCRIPTION OF THE INVENTION

An efficient and rapid method for isolating circulating miRNAs has beendiscovered. As illustrated in the examples, a method of the disclosurecan simultaneously isolate both vesicle-associated and non-vesicleassociated circulating miRNAs. Advantageously, the methods and kits ofthe present disclosure allow for the rapid and specific isolation ofpure preparations of miRNAs with no contamination by other types of RNA.Additionally, the methods and kits disclosed herein allow for isolationof miRNAs in high yield from dilute extracellular fluids. Moreover,methods of the present invention are scalable, allowing miRNA isolationfrom increasing volumes of extracellular biological fluids.

Levels of miRNAs are correlated with disease, including cancer,cardiovascular disease, and in numerous other diseases and developmentalprocesses, including schizophrenia, Alzheimer's disease, immune celldevelopment and modulation of both adaptive and innate immunity, stemcell maintenance and pluripotency, nervous system development, endocrinedisease, including diabetes, development of the pancreas, Fragile XSyndrome, cutaneous wound healing, cell cycle progression, transplantedtissue rejection, hypoxia, skeletal muscle differentiation.Additionally, miRNAs also are expressed by viruses, and target genes ofthose miRNA have been identified. As such, methods and kits of thepresent disclosure can be used to prepare miRNA for assays to diagnosediseases or disease states using a readily available biological sample,such as blood, serum, or plasma.

I. Method

The present disclosure encompasses a method for isolating microRNA(miRNA) from a biological fluid. The method comprises contacting thebiological fluid with a surface active agent and an anti-miRNA-bindingprotein reagent. The surface active agent dissociates biological fluidcomponents and the anti-miRNA-binding protein reagent interacts withmiRNA-binding protein(s) associated with miRNA to formimmunoprecipitated miRNA complexes. The method further comprisesreleasing miRNA from the immunoprecipitated miRNA complexes.

The method disclosed herein specifically isolates miRNAs. As detailed inExample 12 below, other types of small RNAs (such as small nuclear RNAsor small nucleolar RNAs) are not isolated by the disclosed method, andlarger RNA molecules (such as messenger RNAs or ribosomal RNAs) are notisolated by the disclosed method.

(a) Biological Fluid

A method of the present disclosure comprises isolation of extracellularcirculating miRNA in a biological fluid sample obtained from a subject.The term “subject,” as used herein, refers to a human or an animal. Thesubject can be an embryo, a juvenile, or an adult. The subject can bemale or female. Suitable animals include vertebrates such as mammals,birds, reptiles, amphibians, and fish. Examples of suitable mammalsinclude, without limit, rodents, companion animals, livestock, andprimates. Non-limiting examples of rodents include mice, rats, hamsters,gerbils, and guinea pigs. Suitable companion animals include, but arenot limited to, cats, dogs, rabbits, hedgehogs, and ferrets.Non-limiting examples of livestock include horses, goats, sheep, swine,cattle, llamas, and alpacas. Suitable primates include, but are notlimited to, capuchin monkeys, chimpanzees, lemurs, macaques, marmosets,tamarins, spider monkeys, squirrel monkeys, and vervet monkeys.Non-limiting examples of birds include chickens, turkeys, ducks, andgeese. An exemplary subject is a human.

The term “biological fluid” can refer to all biological fluids andexcretions isolated from any given subject. Non-limiting examples of abiological fluid can include blood and fractions thereof, blood serum,blood plasma, urine, excreta, semen, seminal fluid, seminal plasma,prostatic fluid, pre-ejaculatory fluid (Cowper's fluid), pleuraleffusion, tears, saliva, sputum, sweat, biopsy, ascites, cerebrospinalfluid, amniotic fluid, lymph, marrow, cervical secretions, vaginalsecretions, endometrial secretions, gastrointestinal secretions,bronchial secretions, breast secretions, ovarian cyst secretions, tissuefluid, tumor aspirant, and tissue fluid samples. In some embodiments, abiological fluid is blood serum. In other embodiments, a biologicalfluid is blood plasma.

Methods of obtaining a blood plasma or serum sample from a subject arewell known in the art. For instance, venipuncture, with or without acatheter, may be used to collect a blood sample for preparing serum.Methods of preparing plasma and serum from a blood sample are known inthe art. In general, a blood sample is large enough to supply sufficientamounts of plasma or serum to be processed as described further below. Aplasma or serum sample may be processed immediately after collecting thesample. Alternatively, a plasma or serum sample may be frozen for laterprocessing.

A biological fluid sample can be obtained from a subject by freshlycollecting a sample. Alternatively, a biological fluid sample can beobtained from a previously collected and stored sample. For instance,when a biological fluid is blood plasma or serum, a sample can beobtained from a collection of stored and preserved blood samples. Insome embodiments, a sample is obtained by freshly collecting a sample.In other embodiments, a sample is obtained from a previously collectedand stored sample.

In some embodiments, a biological fluid sample is undiluted. In otherembodiments, a biological fluid sample is diluted before isolation ofmiRNA. The degree of dilution may depend on a number of factorsincluding but not limited to the miRNA, the type of biological fluid inthe sample, the subject, the disease condition of the subject, the typeof assay used to measure the miRNA, and the reagents utilized in theassay used to measure the miRNA. In one embodiment, a biological fluidsample is diluted by adding a volume of diluent ranging from about ½ ofthe original sample volume to about 50,000 times the original samplevolume. The diluent may be any fluid that does not interfere with miRNAisolation or other methods used in subsequent processing steps.Non-limiting examples of suitable diluents include deionized water,distilled water, saline solution, Ringer's solution, phosphate bufferedsaline solution, TRIS-buffered saline solution, standard saline citrate,and HEPES-buffered saline.

(b) Surface Active Agent

A biological fluid is contacted with a surface active agent(alternatively referred to as a “surfactant” or “detergent”). As usedherein, the term “surface active agent” can be used to describe anyagent capable of dissociating biological fluid components that cancomprise a circulating miRNA. Non-limiting examples of biological fluidcomponents that can comprise a circulating miRNA include extracellularvesicles such as lipoproteins, exosomes, microvesicles, ectosomes,apoptotic bodies, and other extracellular vesicles.

As will be appreciated by a skilled artisan, any surface active agentcapable of dissociating biological fluid components can be used inmethods of the disclosure, provided that the surface active agent doesnot interfere with formation of an immunoprecipitated miRNA complex ofthe disclosure. For instance, a surface active agent can be an anionicsurface active agent, a cationic surface active agent, a zwitterionicsurface active agent, a non-ionic surface active agent, or combinationsthereof. The identity of the surface active agent of the invention canand will vary, depending upon the identity of the biological fluidcomponents in a biological fluid that may comprise a circulating miRNA,the anti-miRNA-binding protein reagent, and the isolated miRNA.

In some embodiments, a surface active agent is an anionic surface activeagent. Suitable anionic surface active agents include, but are notlimited to, amine dodecylbenzene sulfonate; ammonium capryleth sulfate;ammonium cumenesulfonate; ammonium dihydroxy stearate; ammoniumdodecylbenzene sulfonate; ammonium laureth sulfate; ammonium laureth-12sulfate; ammonium laureth-30 sulfate; ammonium lauryl sarcosinate;ammonium lauryl sulfate; ammonium lauryl sulfosuccinate; ammoniumlignosulfonate; ammonium myreth sulfate; ammonium naphthalene sulfonate;ammonium nonoxynol-20 sulfate; ammonium nonoxynol-30 sulfate; ammoniumnonoxynol-4 sulfate; ammonium nonoxynol-6 sulfate; ammonium nonoxynol-9sulfate; ammonium oleic sulfate; ammonium perfluorooctanoate; ammoniumstearate; ammonium xylenesulfonate; butyl naphthalene sulfonate; butylphosphate; calcium dodecylbenzene sulfonate; calcium stearoyl lactylate;calcium tetrapropylenebenzene sulfonate; capryleth-9 carboxylic acid;cetyl phosphate; cumene sulfonic acid; DEA-cetyl phosphate;DEA-dodecylbenzene sulfonate; DEA-lauryl sulfate; deceth-4 phosphate;diammonium lauryl sulfosuccinate; diammonium stearyl sulfosuccinamate;diamyl sodium sulfosuccinate; dicyclohexyl sodium sulfosuccinate;dihexyl sodium sulfosuccinate; diisobutyl sodium sulfosuccinate;dilaureth-7 citrate; dimethiconol; dinonoxynol-4 phosphate; dioctylammonium sulfosuccinate; dioctyl sodium sulfosuccinate; disodiumcetearyl sulfosuccinamate; disodium cocamido MEA-sulfosuccinate;disodium cocamido PEG-3 sulfosuccinate; disodium deceth-6sulfosuccinate; disodium decyl diphenyl ether disulfonate; disodiumdodecyloxy propyl sulfosuccinamate; disodium isodecyl sulfosuccinate;disodium laneth-5 sulfosuccinate; disodium lauramido DEA-sulfosuccinate;disodium lauramido MEA-sulfosuccinate; disodium laureth sulfosuccinate;disodium lauryl sulfosuccinate; disodium myristamido MEA-sulfosuccinate;disodium oleamido MEA-sulfosuccinate; disodium oleamido PEG-2sulfosuccinate; disodium oleth-3 sulfosuccinate; disodium PEG-4 cocamidoMIPA sulfosuccinate; disodium ricinoleamido MEA-sulfosuccinate; disodiumstearyl sulfosuccinamate; disodium undecylenamido MEA-sulfosuccinate;ditridecyl sodium sulfosuccinate; dodecenylsuccinic anhydride; dodecyldiphenyl ether disulfonic acid; dodecyl diphenyloxide disulfonic acid;dodecylbenzenesulfonic acid; glyceryl dioleate SE; glyceryl distearateSE; glyceryl ricinoleate SE; glyceryl stearate citrate; glycerylstearate SE; glycol stearate SE; hexyl phosphate; isopropyl phosphate;isopropylamine dodecylbenzenesulfonate; isosteareth-2 phosphate;isotrideceth-3 phosphate; isotrideceth-6 phosphate; laureth-1 phosphate;laureth-12 carboxylic acid; laureth-3 phosphate; laureth-4 phosphate;laureth-6 phosphate; laureth-7 citrate; laureth-9 phosphate; laurylphosphate; lithium lauryl sulfate; magnesium laureth sulfate; magnesiumPEG-3 cocamide sulfate; MEA-laureth phosphate; MEA-lauryl sulfate;MIPA-laureth sulfate; MIPA-lauryl sulfate; myristoyl sarcosine;naphthalene-formaldehyde sulfonate; nonoxynol-10 phosphate; nonoxynol-12phosphate; nonoxynol-3 phosphate; nonoxynol-4 phosphate; nonoxynol-4sulfate; nonoxynol-6 phosphate; nonoxynol-7 phosphate; nonoxynol-8phosphate; nonoxynol-9 phosphate; nonyl nonoxynol-10 phosphate; nonylnonoxynol-15 phosphate; nonyl nonoxynol-7 phosphate; oleth-10 carboxylicacid; oleth-10 phosphate; oleth-3 carboxylic acid; oleth-4 phosphate;oleth-5 phosphate; oleth-6 carboxylic acid; oleth-7 phosphate; PEG-2dilaurate SE; PEG-2 dioleate SE; PEG-2 distearate SE; PEG-2 laurate SE;PEG-2 oleate SE; PEG-2 stearate SE; PEG-9 stearamide carboxylic acid;potassium cetyl phosphate; potassium deceth-4 phosphate; potassiumdodecylbenzene sulfonate; potassium isosteareth-2 phosphate; potassiumlauroyl sarcosinate; potassium lauryl sulfate; potassium oleate;potassium oleic sulfate; potassium perfluorooctoate; potassiumricinoleic sulfate; PPG-2 laurate SE; PPG-2 oleate SE; PPG-2 stearateSE; PPG-5-ceteth-10 phosphate; propylene glycol laurate SE; propyleneglycol oleate SE; propylene glycol ricinoleate SE; propylene glycolstearate SE; PVM/MA copolymer; sodium 2-ethylhexyl phosphate; sodium2-ethylhexyl sulfate; sodium a olefin sulfonate; sodium allyloxyhydroxypropyl sulfonate; sodium behenoyl lactylate; sodium butoxyethoxyacetate; sodium butyl naphthalene sulfonate; sodium butyl oleatesulfate; sodium butyl oleate sulfonate; sodium butyl phosphate; sodiumcaproyl lactylate; sodium caprylyl sulfonate; sodium cetyl sulfate;sodium cholate; sodium cumenesulfonate; sodium deceth sulfate; sodiumdecyl diphenyl ether sulfonate; sodium decyl sulfate; sodiumdeoxycholate; sodium dibutyl naphthalene sulfonate; sodiumdidodecylbenzene sulfonate; sodium diisooctyl sulfosuccinate; sodiumdiisopropyl naphthalene sulfonate; sodium dilaureth-7 citrate; sodiumdinonyl sulfosuccinate; sodium dodecyl diphenyl ether disulfonate;sodium dodecyl diphenyloxide disulfonate; sodiumdodecylbenzenesulfonate; sodium glyceryl trioleate sulfate; sodiumhexadecyl diphenyl disulfonate; sodium hexadecyl diphenyloxidedisulfonate; sodium hexyl diphenyloxide disulfonate; sodium isothionate;sodium isodecyl sulfate; sodium isooctyl sulfate; sodium isostearoyllactylate; sodium isotrideceth-15 sulfate; sodium lactate; sodiumlauramido DEA-sulfosuccinate; sodium laureth phosphate; sodium laurethsulfate; sodium laureth sulfosuccinate; sodium laureth-10 phosphate;sodium laureth-11 carboxylate; sodium laureth-12 sulfate; sodiumlaureth-13 acetate; sodium laureth-13 carboxylate; sodium laureth-3carboxylate; sodium laureth-4 carboxylate; sodium laureth-4 phosphate;sodium laureth-6 carboxylate; sodium laureth-7 carboxylate; sodiumlaureth-7 sulfate; sodium laureth-8 sulfate; sodium lauroyl glutamate;sodium lauroyl lactylate; sodium lauroyl lactylate; sodium lauroylmethylaminopropionate; sodium lauroyl sarcosinate; sodium laurylphosphate; sodium lauryl sulfate; sodium lauryl sulfoacetate; sodiumlignate; sodium lignosulfonate; sodium methallyl sulfonate; sodiummethyl lauroyl taurate; sodium methyl myristoyl taurate; sodium methyloleoyl taurate; sodium methyl palmitoyl taurate; sodium methyl stearoyltaurate; sodium methylnaphthalenesulfonate; sodiumm-nitrobenzenesulfonate; sodium myreth sulfate; sodium myristoylglutamate; sodium myristoyl sarcosinate; sodium myristyl sulfate; sodiumnonoxynol sulfate; sodium nonoxynol-10 sulfate; sodium nonoxynol-10sulfosuccinate; sodium nonoxynol-15 sulfate; sodium nonoxynol-4 sulfate;sodium nonoxynol-5 sulfate; sodium nonoxynol-6 phosphate; sodiumnonoxynol-6 sulfate; sodium nonoxynol-8 sulfate; sodium nonoxynol-9phosphate; sodium nonoxynol-9 sulfate; sodium octoxynol-2 ethanesulfonate; sodium octoxynol-3 sulfate; sodium octyl sulfate; sodiumoctylphenoxyethoxyethyl sulfonate; sodium oleic sulfate; sodium oleth-7phosphate; sodium oleyl phosphate; sodium oleyl sulfate; sodium oleylsulfosuccinamate; sodium palmitoyl sarcosinate; sodium phenyl sulfonate;sodium propyl oleate sulfate; sodium stearoyl lactylate; sodium stearylsulfosuccinamate; sodium trideceth sulfate; sodium trideceth-3carboxylate; sodium trideceth-6 carboxylate; sodium trideceth-7carboxylate; sodium tridecyl sulfate; sodium tridecylbenzene sulfonate;sodium xylenesulfonate; stearoyl sarcosine; TEA-lauroyl glutamate;TEA-lauryl sulfate; tetrasodium dicarboxyethyl stearyl sulfosuccinamate;TIPA-laureth sulfate; triceteareth-4 phosphate; triceteth-5 phosphate;trideceth-2 phosphate; trideceth-3 phosphate; trideceth-5 phosphate;tridecyl phosphate; and trilaureth-4 phosphate; and trioctyl phosphate.

In other embodiments, a surface active agent is a cationic surfaceactive agent. Examples of suitable cationic surface active agentsinclude, but are not limited to, alkyltrimethylammonium bromide;benzalkonium chloride; benzalkonium chloride;benzyldimethylhexadecylammonium chloride;benzyldimethyltetradecylammonium chloride; benzyldodecyldimethylammoniumbromide; benzyltrimethylammonium tetrachloroiodate;cetyltrimethylammonium bromide (CTAB); dimethyldioctadecylammoniumbromide; dodecylethyldimethylammonium bromide; dodecyltrimethylammoniumbromide; dodecyltrimethylammonium bromide; dodecyltrimethylammoniumchloride; ethylhexadecyldimethylammonium bromide; Girard's reagent T;hexadecyltrimethylammonium bromide; hexadecyltrimethylammonium bromide;N,N′,N′-polyoxyethylene(10)-N-tallow-1,3-diaminopropane; thonzoniumbromide; and trimethyl(tetradecyl)ammonium bromide.

In yet other embodiments, a surface active agent is a zwitterionicsurface active agent. Suitable zwitterionic surface active agentsinclude, but are not limited to,3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate(CHAPSO); 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate(CHAPS);3-(4-Heptyl)phenyl-3-hydroxypropyl)dimethylammoniopropanesulfonate(C7BzO); 3-(N,N-dimethyloctylammonio) propanesulfonate inner salt(SB3-8); 3-(decyldimethylammonio) propanesulfonate inner salt (SB3-10;caprylyl sulfobetaine); 3-(dodecyldimethylammonio) propanesulfonateinner salt (SB3-12); 3-(N,N-dimethyltetradecylammonio)propanesulfonate(SB3-14); 3-(N,N-dimethylpalmitylammonio) propanesulfonate (SB3-16);3-(N,N-dimethyloctadecylammonio) propanesulfonate (SB3-18);3-[N,N-dimethyl(3-myristoylaminopropyl)ammonio]propanesulfonate(ASB-14). Other suitable zwitterionic detergents, depending on theembodiment, include: acetylated lecithin; apricotamidopropyl betaine;babassuamidopropyl betaine; behenyl betaine; bis 2-hydroxyethyl tallowglycinate; C12-14 alkyl dimethyl betaine; canolamidopropyl betaine;capric/caprylic amidopropyl betaine; capryloamidopropyl betaine; cetylbetaine; cocamidopropyl betaine; cocamidopropyldimethylaminohydroxypropyl hydrolyzed collagen;N-[3-cocamido)-propyl]-N,N-dimethyl betaine, potassium salt;cocamidopropyl hydroxysultaine; cocamidopropyl sulfobetaine;cocaminobutyric acid; cocaminopropionic acid; cocoamphodipropionic acid;coco-betaine; cocodimethylammonium-3-sulfopropylbetaine;cocoiminodiglycinate; cocoiminodipropionate; coco/oleamidopropylbetaine; cocoyl sarcosinamide DEA; DEA-cocoamphodipropionate;dihydroxyethyl tallow glycinate; dimethicone propyl PG-betaine;N,N-dimethyl-N-lauric acid-amidopropyl-N-(3-sulfopropyl)-ammoniumbetaine; N,N-dimethyl-N-myristyl-N-(3-sulfopropyl)-ammonium betaine;N,N-dimethyl-N-palmityl-N-(3-sulfopropyl)-ammonium betaine;N,N-dimethyl-N-stearamidopropyl-N-(3-sulfopropyl)-ammonium betaine;N,N-dimethyl-N-stearyl-N-(3-sulfopropyl)-ammonium betaine;N,N-dimethyl-N-tallow-N-(3-sulfopropyl)-ammonium betaine; disodiumcaproamphodiacetate; disodium caproamphodipropionate; disodiumcapryloamphodiacetate; disodium capryloamphodipropionate; disodiumcocoamphodiacetate; disodium cocoamphodipropionate; disodiumisostearoamphodipropionate; disodium laureth-5 carboxyamphodiacetate;disodium lauriminodipropionate; disodium lauroamphodiacetate; disodiumlauroamphodipropionate; disodium octyl b-iminodipropionate; disodiumoleoamphodiacetate; disodium oleoamphodipropionate; disodiumPPG-2-isodeceth-7 carboxyamphodiacetate; disodium soyamphodiacetate;disodium stearoamphodiacetate; disodium tallamphodipropionate; disodiumtallowamphodiacetate; disodium tallowiminodipropionate; disodiumwheatgermamphodiacetate;N,N-distearyl-N-methyl-N-(3-sulfopropyl)-ammonium betaine;erucamidopropyl hydroxysultaine; ethylhexyl dipropionate; ethylhydroxymethyl oleyl oxazoline; ethyl PEG-15 cocamine sulfate;hydrogenated lecithin; hydrolyzed protein; isostearamidopropyl betaine;lauramidopropyl betaine; lauramidopropyl dimethyl betaine;lauraminopropionic acid; lauroamphodipropionic acid; lauroyl lysine;lauryl betaine; lauryl hydroxysultaine; lauryl sultaine;linoleamidopropyl betaine; lysolecithin; milk lipid amidopropyl betaine;myristamidopropyl betaine; octyl dipropionate; octyliminodipropionate;oleamidopropyl betaine; oleyl betaine; 4,4(5H)-oxazoledimethanol,2-(heptadecenyl)-; palmitamidopropyl betaine; palmitamine oxide;ricinoleamidopropyl betaine; ricinoleamidopropyl betaine/IPDI copolymer;sesamidopropyl betaine; sodium C12-15 alkoxypropyl iminodipropionate;sodium caproamphoacetate; sodium capryloamphoacetate; sodiumcapryloamphohydroxypropyl sulfonate; sodium capryloamphopropionate;sodium carboxymethyl tallow polypropylamine; sodium cocaminopropionate;sodium cocoamphoacetate; sodium cocoamphohydroxypropyl sulfonate; sodiumcocoamphopropionate; sodium dicarboxyethyl cocophosphoethyl imidazoline;sodium hydrogenated tallow dimethyl glycinate; sodiumisostearoamphopropionate; sodium lauriminodipropionate; sodiumlauroamphoacetate; sodium oleoamphohydroxypropylsulfonate; sodiumoleoamphopropionate; sodium stearoamphoacetate; sodiumtallamphopropionate; soyamidopropyl betaine; stearyl betaine;tallowamidopropyl hydroxysultaine; tallowamphopolycarboxypropionic acid;trisodium lauroampho PG-acetate phosphate chloride; undecylenamidopropylbetaine; and wheat germamidopropyl betaine.

In other embodiments, a surface active agent is preferably a non-ionicsurface active agent. Examples of suitable nonionic surface activeagents include, but are not limited to, polyoxyethylene (10) cetyl ether(BRIJ® 56); polyoxyethylene (20) cetyl ether (BRIJ® 58);polyoxyethyleneglycol dodecyl ether (BRIJ® 35); polyoxyethylene (9)p-t-octyl phenol (NONIDET™ P-40); polyoxyethylene (4-5) p-t-octyl phenol(TRITON™ X-45); polyoxyethylene (7-8) p-t-octyl phenol (TRITON™ X-114);polyoxyethylene (9-10) p-t-octyl phenol (TRITON™ X-100); polyoxyethylene(9-10) nonylphenol (TRITON™ N-101); polyoxyethylene (20) sorbitolmonolaurate (TWEEN® 20); polyoxyethylene (20) sorbitol monopalmitate(TWEEN® 40); polyoxyethylene (20) sorbitol monooleate (TWEEN® 80);dimethyldecylphosphine oxide (APO-10); dimethyldodecylphosphine oxide(APO-12); cyclohexyl-n-ethyl-β-D-maltoside;cyclohexyl-n-hexyl-β-D-maltoside; cyclohexyl-n-methyl-β-maltoside;n-decanoylsucrose; n-decyl-β-D-glucopyranoside;n-decyl-β-maltopyranoside; n-decyl-β-D-thiomaltoside; n-dodecanoylsucrose; decaethylene glycol monododecyl ether;N-decanoyl-N-methylglucamine; n-decyl α-D-glucopyranoside; decylβ-D-maltopyranoside; n-dodecanoyl-N-methylglucamide; n-dodecylα-D-maltoside; n-dodecyl β-D-maltoside; heptane-1,2,3-triol;heptaethylene glycol monodecyl ether; heptaethylene glycol monododecylether; heptaethylene glycol monotetradecyl ether; n-hexadecylβ-D-maltoside; hexaethylene glycol monododecyl ether; hexaethyleneglycol monohexadecyl ether; hexaethylene glycol monooctadecyl ether;hexaethylene glycol monotetradecyl ether;methyl-6-O-(N-heptylcarbamoyl)-α-D-glucopyranoside; nonaethylene glycolmonododecyl ether; N-nonanoyl-N-methylglucamine;N-nonanoyl-N-methylglucamine; octaethylene glycol monodecyl ether;octaethylene glycol monododecyl ether; octaethylene glycol monohexadecylether; octaethylene glycol monooctadecyl ether; octaethylene glycolmonotetradecyl ether; octyl-β-glucoside; octyl-β-thioglucoside;octyl-β-D-glucopyranoside; octyl-β-D-1-thioglucopyranoside;pentaethylene glycol monodecyl ether; pentaethylene glycol monododecylether; pentaethylene glycol monohexadecyl ether; pentaethylene glycolmonohexyl ether; pentaethylene glycol monooctadecyl ether; pentaethyleneglycol monooctyl ether; polyethylene glycol diglycidyl ether;polyethylene glycol ether; polyoxyethylene 10 tridecyl ether;polyoxyethylene (100) stearate; polyoxyethylene (20) isohexadecyl ether;polyoxyethylene (20) oleyl ether; polyoxyethylene (40) stearate;polyoxyethylene (50) stearate; polyoxyethylene (8) stearate;polyoxyethylene bis(imidazolyl carbonyl); polyoxyethylene (25) propyleneglycol stearate; saponin from Quillaja bark; tetradecyl-β-D-maltoside;tetraethylene glycol monodecyl ether; tetraethylene glycol monododecylether; tetraethylene glycol monotetradecyl ether; triethylene glycolmonodecyl ether; triethylene glycol monododecyl ether; triethyleneglycol monohexadecyl ether; triethylene glycol monooctyl ether;triethylene glycol monotetradecyl ether; tyloxapol; n-undecylβ-D-glucopyranoside, (octylphenoxy)polyethoxyethanol (IGEPAL® CA-630);polyoxyethylene (5) nonylphenylether (IGEPAL® CO-520); andpolyoxyethylene (150) dinonylphenyl ether (IGEPAL® DM-970). In oneembodiment, a surface active agent is polyoxyethylene (5)nonylphenylether (IGEPAL® CO-520). In another embodiment, a surfaceactive agent is polyoxyethylene (150) dinonylphenyl ether (IGEPAL®DM-970). In one embodiment, a surface active agent is preferably(octylphenoxy) polyethoxyethanol (IGEPAL® CA-630).

As will be appreciated by a skilled artisan, the amount of surfaceactive agent added to the biological fluid can and will vary dependingupon the identity of the biological fluid components in a biologicalfluid that may comprise a circulating miRNA. In some embodiments, thefinal concentration of surface active agent in the biological fluid canrange from about 0.001 to about 10%. In one embodiment, theconcentration of surface active agent can range from about 0.001 toabout 0.01%. In another embodiment, the concentration of surface activeagent can range from about 0.01% to about 0.1%. In yet anotherembodiment, the concentration of surface active agent can range fromabout 0.1% to about 1%. In another embodiment, the concentration ofsurface active agent can range from about 1% to about 5%. In anadditional embodiment, the concentration of surface active agent canrange from about 5% to about 10%.

(c) Anti-miRNA-Binding Protein Reagent

A biological fluid is contacted with an anti-miRNA-binding proteinreagent. An anti-miRNA-binding protein reagent can be any agent capableof binding a miRNA-binding protein associated with circulating miRNAs. AmiRNA-binding protein associated with circulating miRNAs can bind amiRNA directly, or can be indirectly associated with an RNA-proteincomplex comprising miRNA. Non-limiting examples of miRNA-bindingproteins that can be associated with circulating miRNAs can includeArgonaut, Dicer, human immunodeficiency virus (HIV) transactivatingresponse RNA binding protein (TRBP), protein activator of the interferoninduced protein kinase (PACT), the SMN complex, fragile X mentalretardation protein (FMRP), Tudor staphylococcalnuclease-domain-containing protein (Tudor-SN), the putative DNA helicaseMOV10, and the RNA recognition motif containing protein TNRC6B, or othercomponents of the RISC complex or that may associate transiently orpermanently with the RISC complex.

In some embodiments, a biological fluid is preferably contacted with ananti-Argonaut reagent. Non-limiting examples of an Argonaut protein caninclude Ago1, Ago2, Ago3, and Ago4. In one embodiment, a biologicalfluid is contacted with an anti-Ago1 reagent. In another embodiment, abiological fluid is contacted with an anti-Ago2 reagent. In yet anotherembodiment, a biological fluid is contacted with an anti-Ago3 reagent.In an additional embodiment, a biological fluid is contacted with ananti-Ago4 reagent. In another embodiment, a biological fluid ispreferably contacted with a reagent capable of binding more than oneArgonaut protein. For example, a biological fluid can be contacted withan anti-Ago1 and an anti-Ago2 reagent. In still another embodiment, abiological fluid is preferably contacted with a reagent capable ofbinding Ago1, Ago2, Ago3, and Ago4.

An anti-miRNA-binding protein reagent can be an epitope binding agent.Non-limiting examples of suitable epitope binding agents, depending uponthe target molecule, include agents selected from the group consistingof an aptamer, an antibody, an antibody fragment, a double-stranded DNAsequence, modified nucleic acids, nucleic acid mimics, a ligand, aligand fragment, a receptor, a receptor fragment, a polypeptide, apeptide, a coenzyme, a coregulator, an allosteric molecule, and an ion.

In some embodiments, an epitope binding agent is an antibody.Non-limiting examples of antibodies that can be used include polyclonalantibodies, ascites, Fab fragments, Fab′ fragments, monoclonalantibodies, single chain antibodies, single domain antibodies, humanizedantibodies, and other fragments that contain the epitope binding site ofthe antibody.

In some embodiments, a biological fluid is contacted with ananti-Argonaut antibody. In one embodiment, a biological fluid iscontacted with an anti-Ago1 antibody. In another embodiment, abiological fluid is contacted with an anti-Ago2 antibody. In yet anotherembodiment, a biological fluid is contacted with an anti-Ago3 antibody.In another embodiment, a biological fluid is contacted with an anti-Ago4antibody. In an additional embodiment, a biological fluid is contactedwith two anti-Ago antibodies chosen from anti-Ago1, anti-Ago2,anti-Ago3, or antiAgo4 antibodies. For example, a biological fluid iscontacted with anti-Ago1 and anti-Ago2 antibodies. In a furtherembodiment, a biological fluid is contacted with three anti-Agoantibodies chosen from anti-Ago1, anti-Ago2, anti-Ago3, or antiAgo4. Inyet another embodiment, a biological fluid is contacted with all fouranti-Ago antibodies. In a further embodiment, a biological fluid iscontacted with an antibody capable of recognizing more than one Argonautprotein. Such antibodies may recognize one, two, three, or four Argonautproteins. In one embodiment, a biological fluid is contacted with ananti-Argonaut antibody capable of recognizing all four human Argonautproteins.

Contacting a biological fluid with an anti-miRNA-binding protein reagentof the disclosure forms immunoprecipitated miRNA complexes. As such, ananti-miRNA-binding protein reagent is normally attached to a solidsupport to form immunoprecipitated miRNA complexes when a biologicalfluid is contacted with the immobilized anti-miRNA-binding proteinreagent. The solid support can be a material that can be modified tocontain discrete individual sites appropriate for the attachment orassociation of an anti-miRNA-binding protein reagent. Non-limitingexamples of solid support materials include glass, modified orfunctionalized glass, plastics including acrylics, polystyrene andcopolymers of styrene and other materials, polypropylene, polyethylene,polybutylene, polyurethanes, or TeflonJ, nylon, nitrocellulose,polysaccharides, resins, silica or silica-based materials includingsilicon and modified silicon, carbon, metals, inorganic glasses andplastics. The size and shape of the solid support can vary withoutdeparting from the scope of the invention. A solid support can beplanar, a solid support can be a well, i.e., a 364 well plate, oralternatively, a solid support can be a bead or a slide. In someembodiments, a solid support is a well of a multiwall plate. In otherembodiments, a solid support is an inner surface of a pipette tip. Inyet other embodiments, a solid support is preferably a bead. In someembodiments, a solid support is preferably a magnetic bead.

An anti-miRNA-binding protein reagent can be attached to a solid supportin a wide variety of ways, as will be appreciated by those in the art.An anti-miRNA-binding protein reagent and a solid support can bederivatized with chemical functional groups for subsequent attachment ofthe two. For example, a solid support can be derivatized with a chemicalfunctional group including, but not limited to, amino groups, carboxylgroups, oxo groups or thiol groups. Using these functional groups, ananti-miRNA-binding protein reagent can be attached using functionalgroups either directly, or indirectly using linkers. Alternatively,anti-miRNA-binding protein reagent can also be attached to the solidsupport non-covalently. For example, a biotinylated anti-miRNA-bindingprotein reagent can be prepared, which can bind to a solid supportcovalently coated with streptavidin, resulting in attachment. Additionalmethods of attaching an anti-miRNA-binding protein reagent to a solidsupport are well known in the art, and may be as described in publishedlaboratory manuals such as in “Current Protocols in Molecular Biology”Ausubel et al., John Wiley & Sons, New York, 2003 or “Molecular Cloning:A Laboratory Manual” Sambrook & Russell, Cold Spring Harbor Press, ColdSpring Harbor, N.Y., 3rd edition, 2001. In some embodiments, abiotinylated anti-miRNA-binding protein reagent is prepared, which canbind to a bead solid support covalently coated with streptavidin,resulting in attachment. As described in Section I(d) below, ananti-miRNA-binding protein reagent can be attached to a solid supportbefore contacting a biological fluid in a method of the disclosure.Alternatively, a biological fluid of the disclosure can be contactedsimultaneously with an anti-miRNA-binding protein reagent and a solidsupport, where the anti-miRNA-binding protein reagent attaches to thesolid support. A biological fluid of the disclosure can also becontacted with an anti-miRNA-binding protein reagent before contactingthe biological fluid with a solid support, where the anti-miRNA-bindingprotein reagent attaches to the solid support. As will be appreciated bya skilled artisan, the amount and concentration of anti-miRNA-bindingprotein reagent can and will vary depending upon the identity of theanti-miRNA-binding protein reagent, the volume of biological fluid used,the concentration of a miRNA in the biological fluid, and themiRNA-binding protein among other factors, and may be determinedexperimentally. When an anti-miRNA-binding protein reagent is a purifiedantibody, about 0.5 to about 10 μg of antibody can be used for each 0.2ml plasma or serum sample.

(d) Contacting Biological Fluid and Isolating miRNA

In a method of the disclosure, a biological fluid is contacted with asurface active agent and an anti-miRNA-binding protein reagent. As willbe appreciated by a skilled artisan, a biological fluid can be contactedwith a variety of other agents without departing from the scope of theinvention. For instance, a biological fluid can be contacted with athiol-reducing agent to block the formation of disulfide bonds andinhibit ribonuclease activity during miRNA isolation. Suitablethiol-reducing agents include dithiothreitol (DTT), 2-mercaptoethanol,2-mercaptoethylamine, and tris(carboxyethyl) phosphine (TCEP). Abiological fluid can also be contacted with an antifoaming agent.Examples of antifoaming agents include Antifoam 204 and Antifoam O-30,Antifoam A, Antifoam B, Antifoam C, Antifoam Y-30, and Sag 471. Abiological fluid can also be contacted with RNA and protein degradationinhibitors to preserve miRNA and miRNA-protein complexes.

In some embodiments a buffering agent can be used to maintain a pHsuitable for isolating miRNAs. By way of non-limiting example, bufferingagents may include, but are not limited to, trizma acetate, EDTA, tris,glycine, and citrate.

In some embodiments, a method of the disclosure comprises contacting abiological fluid with a surface active agent to dissociate biologicalfluid components before contacting the biological fluid with ananti-miRNA-binding protein reagent to form immunoprecipitated miRNAcomplexes. In other embodiments, a biological fluid is contacted with asurface active agent and an anti-miRNA-binding protein reagentsimultaneously.

In some embodiments, an undiluted sample of biological fluid iscontacted with a surface active agent and an anti-miRNA-binding proteinreagent. In other embodiments, a biological fluid is diluted beforecontacting with a surface active agent and an anti-miRNA-binding proteinreagent. Dilution of a biological fluid may be as described in sectionI(a) above.

Contact between a biological fluid, a surface active agent, and ananti-miRNA-binding protein reagent generally comprises a period ofincubation to allow formation of immunoprecipitated miRNA complexes. Abiological fluid can be contacted with a surface active agent and ananti-miRNA-binding protein reagent and incubated for about 1, 5, 10, 15,30, 45, 60, 90, 120, 240 or 480 minutes or longer. In some embodiments,a biological fluid is contacted with a surface active agent and ananti-miRNA-binding protein reagent and incubated for about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or about 15 minutes. In otherembodiments, a biological fluid is contacted with a surface active agentand an anti-miRNA-binding protein reagent and incubated for about 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29 or about 30 minutes. In yet other embodiments, a biological fluid iscontacted with a surface active agent and an anti-miRNA-binding proteinreagent and incubated for about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80 85, or about 90 minutes. In other embodiments, a biologicalfluid is contacted with a surface active agent and an anti-miRNA-bindingprotein reagent and incubated for about 90, 120, 240 or 480 minutes orlonger. In one embodiment, a biological fluid is preferably contactedwith a surface active agent and an anti-miRNA-binding protein reagentand incubated for about 20, 25, 30, 35, 40, 45, 50, 55, or about 60minutes.

A biological fluid can be contacted with a surface active agent and ananti-miRNA-binding protein reagent at a temperature of about 0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or about 30° C. or more. In someembodiments, a biological fluid is contacted with a surface active agentand an anti-miRNA-binding protein reagent at a temperature of about 0,1, 2, 3, 4, 5, or about 6° C. In other embodiments, a biological fluidis contacted with a surface active agent and an anti-miRNA-bindingprotein reagent at a temperature of about 5, 6, 7, 8, 9, 10, 11, 12, 13,14, or about 15° C. In other embodiments, a biological fluid iscontacted with a surface active agent and an anti-miRNA-binding proteinreagent at a temperature of about 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, or about 25° C. In yet other embodiments, abiological fluid is contacted with a surface active agent and ananti-miRNA-binding protein reagent at a temperature of about 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or about 30° C.

Typically, a biological fluid is contacted with a surface active agentand an anti-miRNA-binding protein reagent under agitation. Additionally,a biological fluid can generally be removed to isolateimmunoprecipitated miRNA complexes after forming the complexes, andimmunoprecipitated miRNA complexes washed.

(e) Releasing miRNA

According to a method of the disclosure, miRNA is released fromimmunoprecipitated miRNA complexes. Methods of releasing a nucleic acidsuch as a miRNA from a protein complex are well known in the art and mayinclude protease digestion, and denaturation of proteins in a nucleicacid-protein complex. In some embodiments, miRNA is released fromimmunoprecipitated miRNA complexes by protein denaturation. Forinstance, miRNA can be released from immunoprecipitated miRNA complexesby combining immunoprecipitated miRNA complexes with a guanidiniumthiocyanate-phenol-chloroform solution. Released miRNA can then bepurified by precipitation or using spin column chromatography.

In other embodiments, miRNA is preferably released fromimmunoprecipitated miRNA complexes by protease digestion. The terms“protease”, “proteinase”, and “peptidase” are used interchangeablyherein and refer to the group of enzymes that catalyze the hydrolysis ofcovalent peptidic bonds. Protease enzymes are well known in the art andmay include acid proteases and serine proteases. In some embodiments, aprotease that can be used to release miRNA in a method of the disclosureis an acid protease. In one embodiment, an acid protease that may beused to release miRNA in a method of the disclosure is pepsin.

In other embodiments, a protease that may be used to release miRNA in amethod of the disclosure is an acid protease. Six clans of serineproteases have been identified, the two largest of which are thechymotrypsin-like and the subtilisin-like clans. A large number ofsubtilases are known. Some of the subtilases which have been extensivelystudied include those obtained from various species of Bacillusincluding subtilisin DY, subtilisin Carlsberg, subtilisin BPN′ (alsocalled nagarse), mesentericopeptidase, as well as proteinase K which isobtained from Tritirachium album Limber, and thermitase which isobtained from Thermoactinomyces vulgaris. In certain embodiments of thepresent invention, proteinase K is preferred as a protease enzyme. Otherprotease enzymes, however, can also be used in certain embodiments, suchas, for example, nagarse. The protease enzyme can thus be any of anumber of proteases that produce at least a partial breakdown ofproteins in immunoprecipitated miRNA complexes such that miRNA isreleased. In some embodiments, a protease that may be used to releasemiRNA in a method of the disclosure is preferably protease K.

In essence, miRNA is released from immunoprecipitated miRNA complexes bycontacting complexes with a protease enzyme. As will be appreciated by askilled artisan, the amount of protease used to release miRNA can andwill vary depending on the protease, the abundance of immunoprecipitatedmiRNA complexes, the temperature during protease digestion, the bufferconditions used for digestion and the duration of digestion, among otherfactors. In general, immunoprecipitated miRNA complexes can be contactedwith about 0.3 units of enzyme activity to about 30 units of enzymeactivity. In certain embodiments, the amount of protease contacted withimmunoprecipitated miRNA complexes can range from about 0.3 to about 1unit, from about 1 to about 3 units, from about 3 units to about 10units, or from about 10 units to about 30 units.

In some embodiments, using protease digestion at room temperature asdescribed in Example 1. As used herein, the term “room temperature” isused to describe a temperature of about 10° C. to about 30° C.

Immunoprecipitated miRNA complexes can be incubated with a protease forabout 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19 20, 25, or about 30 minutes or longer. In some embodiments,immunoprecipitated miRNA complexes are incubated with a protease forabout 0.5, 1, 2, 3, 4, or about 5 minutes. In other embodiments,immunoprecipitated miRNA complexes are incubated with a protease forabout 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or about 15 minutes. In yetother embodiments, immunoprecipitated miRNA complexes are incubated witha protease for about 15, 16, 17, 18, 19 20, 25, or about 30 minutes orlonger.

Released miRNA may be appropriate for downstream use without furtherpurification. Alternatively, released miRNA may be further purified fordownstream uses. Methods of nucleic acid purification, such as spincolumn chromatography or filtration techniques, are well known in theart, e.g., according to methods described in published laboratorymanuals such as in “Current Protocols in Molecular Biology” Ausubel etal., John Wiley & Sons, New York, 2003 or “Molecular Cloning: ALaboratory Manual” Sambrook & Russell, Cold Spring Harbor Press, ColdSpring Harbor, N.Y., 3rd edition, 2001.

The downstream use of released miRNA may vary. Non-limiting uses ofreleased miRNA include quantitative real-time PCR, microarray analysis,sequencing, restriction fragment length polymorphism (RFLP) analysis,single nucleotide polymorphism (SNP) analysis, microsatellite analysis,short tandem repeat (STR) analysis, and comparative genomichybridization (CGH).

II. Kits

The invention further provides kits comprising surface active agents,anti-miRNA-binding protein reagents, and other reagents that can be usedin a method of the disclosure. In some embodiments, a kit is providedfor isolating miRNA from a biological fluid, which kit includes ananti-miRNA-binding protein reagent and a surface-acting agent.Anti-miRNA-binding protein reagents and surface active agents can be asdescribed in section (I) above. In some embodiments, ananti-miRNA-binding protein reagent in a kit is an anti-Ago antibodyattached to a solid support. In certain embodiments, a solid support canbe a bead, a magnetic bead, or a well of a multiwall plate. In stillother embodiments, a solid support can be an inner surface of a pipettetip. In some embodiments, a surface-acting agent in a kit is IGEPAL. Akit may further comprise a means for releasing miRNA fromimmunoprecipitated miRNA complexes. In some embodiments, a kit comprisesa protease, e.g., protease K, for releasing miRNA fromimmunoprecipitated miRNA complexes.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

When introducing elements of the present disclosure or the preferredaspects(s) thereof, the articles “a”, “an”, “the” and “said” areintended to mean that there are one or more of the elements. The terms“comprising”, “including” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements.

As used herein, “microRNA” or “miRNA” means a small, noncoding RNAsequence of 5 to 40 nucleotides in length that can be detected in abiological specimen. Some miRNAs are derived from hairpin precursorsprocessed, for example, by the enzyme DICER to a mature species, forexample, about 18-25 nucleotides, preferably 21-23 nucleotides. MicroRNAvariants are common, for example, among different animal species. Inaddition, variation at the 5′ and 3′ ends of miRNAs are common, and canbe the result of imprecise cleavage by enzymes such as DICER duringmaturation. These variants demonstrate a scope of acceptable variationin the sequence of the miRNAs that does not impair function or theability to detect the miRNA(s). Another type of variant is post-Dicerprocessing addition of non-templated nucleotide(s) to the 3′ end of themiRNA (these are non-templated because they do not match the humangenome). The most common variants are the miRNA sequence with an extra Aor U added to the 3′ end.

As used herein, the term “biological fluid” or “body fluid” can be usedinterchangeably and refer to a fluid isolated from a subject.

The terms “biological fluid”, “biological fluid sample”, or “biologicalsample” can be used interchangeably and refer to all biological fluidsand excretions isolated from any given subject. In the context of theinvention such samples include, but are not limited to, blood andfractions thereof, blood serum, blood plasma, urine, excreta, semen,seminal fluid, seminal plasma, prostatic fluid, pre-ejaculatory fluid(Cowper's fluid), pleural effusion, tears, saliva, sputum, sweat,biopsy, ascites, cerebrospinal fluid, amniotic fluid, lymph, marrow,cervical secretions, vaginal secretions, endometrial secretions,gastrointestinal secretions, bronchial secretions, breast secretions,ovarian cyst secretions, and tissue fluid samples.

An “isolated” polynucleotide is a nucleic acid molecule that isidentified and separated from at least one contaminant with which it isordinarily associated in its natural source. An isolated nucleic acidmolecule is other than in the form or setting in which it is found innature. Isolated nucleic acid molecules therefore are distinguished fromthe specific nucleic acid molecule as it exists in natural cells.

As various changes could be made in the above-described animals, cellsand methods without departing from the scope of the invention, it isintended that all matter contained in the above description and in theexamples given below, shall be interpreted as illustrative and not in alimiting sense.

EXAMPLES

The following examples are included to demonstrate the disclosure. Itshould be appreciated by those of skill in the art that the techniquesdisclosed in the following examples represent techniques discovered bythe inventors to function well in the practice of the disclosure. Thoseof skill in the art should, however, in light of the present disclosure,appreciate that many changes could be made in the disclosure and stillobtain a like or similar result without departing from the spirit andscope of the disclosure, therefore all matter set forth is to beinterpreted as illustrative and not in a limiting sense.

Example 1: General miRNA Isolation Protocol

A representative protocol used to isolate circulating miRNAs comprisesperforming RNA immunoprecipitation (RIP) in the presence of a detergentto release vesicle-associated miRNAs. In this protocol, miRNAs areseparated from other cellular components such as other RNAs, plasmaproteins, etc. without the use of phenol, chaotropes, or columnpurification, and may be completed in 40-70 minutes.

The protocol consists of three steps:

1) Plasma components are treated with a detergent,

2) miRNA/protein complexes are immunoprecipitated, and

3) miRNA is released from immunoprecipitated miRNA complexes.

Protein A (Sigma-Aldrich GE28-9670-56), Protein G (Sigma-AldrichGE28-9670-66), or Streptavidin (Sigma-Aldrich GE28-9857-38) beads werecoated with anti-Ago antibody by transferring 20 μl of magnetic beads(10% slurry) to 0.1 ml RIP wash buffer (50 mM Tris-HCl, pH 7.4, 0.05%IGEPAL® CA-630), washing the beads with RIP wash buffer once, and usinga magnetic stand for separating the beads from the solution. The washedmagnetic beads were then resuspended in 0.1 ml RIP wash buffer beforeadding 2.5-10 μg unbiotinylated or biotinylated anti-Ago (Sigma-AldrichSAB4800048), anti-Ago2 (Sigma-Aldrich SAB4200085), or anti-Ago1(Sigma-Aldrich SAB4200084) antibody. The beads and antibody wereincubated with rotation at room temperature for about 30 minutes. Thebeads were then separated from the solution using a magnetic stand. Theantibody beads were then washed twice with 0.5 ml RIP wash buffer.

In the first step of the miRNA isolation process, 0.2 ml plasma, 8 μl25% IGEPAL CA-630 (Sigma-Aldrich I8896; 40 μl 25% IGEPAL per ml plasmato produce a final concentration of 1%), 2 μl protease inhibitorcocktail (PIC; Sigma-Aldrich P8340; 10 μl/ml plasma), and 0.8 μl RNaseinhibitor (Sigma-Aldrich R1158; 4 μl/ml plasma) were added to theprepared Ago antibody beads. Alternatively, plasma may be treated withdetergent and inhibitors while preparing beads, and pre-treated plasmaadded subsequently to the antibody beads.

In the second step, miRNA/protein complexes were immunoprecipitated byincubating the sample at room temperature for 1 hr or 4° C. overnightwith rotation. The beads were washed 5× with 1 ml wash RIP buffer, andcollected using the magnetic stand to separate the beads from thesupernatant. The beads can be centrifuged briefly and returned to themagnetic stand to remove residual supernatant.

In the third step, precipitated miRNA associated with antibody beads wasreleased from the protein complex and the beads by extraction with TRIReagent®BD or QIAzol lysis reagent followed by isopropanol precipitationwith ammonium acetate and linear acrylamide, as described in theTechnical Bulletin for Sigma-Aldrich Imprint RNA Immunoprecipitation Kit(RIP), or purified with Qiagen's miRNeasy Serum/Plasma Kit.Alternatively, and preferably, miRNA was released by proteinase Kdigestion. Twenty μl proteinase K mix (14 μl water, 2 μl 10× proteinaseK release buffer, and 4 μl P4850 proteinase K) was added to the beadsfrom step two, and incubated at room temperature for 10 minutes onvortex genie 2, setting 4. The 10× proteinase K release buffer comprises100 mM Tris, pH 8.0, 15 mM MgCl₂, 500 mM KCl, 100 mM DTT, and 1% IGEPAL.Immediately after incubation, the beads were removed by placing on amagnetic stand and transferring the supernatant comprising free miRNA toa fresh tube. Proteinase K in the supernatant was then inactivated byincubating the sample at 95° C. for 5 minutes. Specific miRNAs weredetected with Sigma-Aldrich's MystiCq RT-qPCR assays using 5 μl of eachmiRNA preparation per 10 μl polyA-tailing reaction. Synthetic miRNAs,i.e., single-stranded RNA with the same sequence as mature miRNAs listedin miRBase, were diluted in 0.02 mg/ml linear acrylamide to known copynumber based on absorbance of stock solutions at 260 nm, assayed inparallel with miRNAs prepared from plasma, and used as standards forabsolute quantitation.

Example 2: Immunoprecipitation of miRNAs from Plasma is More EfficientThan Tri Reagent Alone

The efficiency of miRNA isolation from plasma using RNAimmunoprecipitation (RIP) was compared to miRNA isolation using TRIReagent®BD (Sigma-Aldrich). TRI Reagent®BD is a reagent for use in thesimultaneous isolation of RNA, DNA and protein from blood derivativessuch as serum, plasma or whole blood.

Isolation of miRNA using TRI Reagent®BD was according to themanufacturer's instructions. In short, 0.2 ml plasma was mixed with TRIReagent®BD, and miRNA was extracted using chloroform for phaseseparation before isopropanol precipitation in the presence of ammoniumacetate and linear acrylamide, and washing of RNA for analysis.Isolation of miRNAs using RIP was performed with 2.5 μg anti-Ago2antibody bound to 20 μl Protein A magnetic beads. miRNA was recoveredfrom the beads by extraction with TRI Reagent®BD and isopropanolprecipitation in the presence of ammonium acetate and linear acrylamide,as for the direct plasma extraction.

The level of let-7a-5p, miR23a-3p, miR191-5p, miR142-3p, and miR451amiRNAs in the prepared samples was determined by quantitative, real-timeRT-PCR. RIP of miRNA from plasma was about 5 to about 600 fold moreefficient than TRI Reagent®BD (FIG. 1).

Example 3. RIP Yield Similar to Yield From a Commercial Kit

The efficiency of miRNA isolation from plasma using RNAimmunoprecipitation (RIP) was compared to miRNA isolation using Qiagen'smiRNeasy Serum/Plasma Kit (Qiagen). Qiagen miRNeasy employs spin columnscomprising silica resin that selectively binds DNA or RNA, and isrecommended for miRNA isolation from ≤0.2 ml serum or plasma.

Isolation of miRNA using Qiagen miRNeasy Serum/Plasma Kit was accordingto the manufacturer's instructions. In short, 0.2 ml plasma was mixedwith QIAzol reagent, and miRNA was purified from the aqueous layer usingthe provided spin columns. Isolation of miRNAs using RIP was performedwith 20 μl Protein A magnetic beads to which 2.5 μg anti-Ago2 antibodywas bound. miRNAs were released from the beads with QIAzol reagent andpurified with the Qiagen kit, as for the direct plasma extraction.

The level of let-7a, miR23a, miR191, miR142, and miR451a miRNAs in theprepared samples was determined by quantitative, real-time RT-PCR. Yieldof miRNAs using RIP was similar to miRNA yield using the Qiagen kitalone (FIG. 2).

Example 4. Comparing RIP Using Biotinylated and Non-BiotinylatedAnti-Ago Antibody and Streptavidin Beads

Protein A and Protein G beads both bind human IgG, which is extremelyabundant in plasma. To avoid co-isolating IgG, anti-Ago (clone 2A8) andanti-Ago2 (clone 11A9) antibodies were biotinylated with Pierce EZ-LinkSulfo-NHS-LC-LC-Biotin (Thermo Scientific) for RIP with streptavidinbeads. Ago-RIP was performed using 2.5 μg of the biotinylated anti-Ago2(b-Ago2) or anti-Ago (b-Ago) antibody and 20 μl streptavidin magneticbeads, or with 2.5 μg of non-biotinylated anti-Ago2 antibody and 20 μlProtein A beads. RIP with biotinylated anti-Ago2 (b-Ago2) antibody andstreptavidin beads gave the same yield of miRNAs as RIP with anti-Ago2antibody with Protein A beads (see FIG. 3). RIP with biotinylatedanti-Ago gave significantly lower miRNA yields, as they had withunbiotinylated anti-Ago and Protein A beads.

Example 5. Heat Release of miRNA Isolated Using RIP Negatively AffectsYield

Heating in nuclease-free water was tested as a means to release miRNAsfollowing RIP. Ago-RIP was performed with 0.2 ml plasma and 2.5 μg ofeither unbiotinylated anti-Ago or anti-Ago2 antibody on Protein Amagnetic beads, or biotinylated anti-Ago or anti-Ago2 antibody onstreptavidin magnetic beads. Fourteen μl of nuclease-free water wasadded to the beads, and these mixtures were heated at 40°, 50°, or 60°C. for 2 minutes before removing the beads. RIP followed by heat releasewas compared with RIP followed by miRNA purification with QiagenmiRNeasy Serum/Plasma Kit, and miRNAs purified directly from plasma withthe Qiagen kit. Synthetic cel-miR-39-3p (1.4e8 copies) was spiked inafter QIAzol addition for Qiagen preps or in the water added to post-RIPbeads.

Synthetic cel-miR-39-3p spike-in was undetected after 2 minutes at 60°C. with RIP product on beads (FIG. 4). There was also no endogenousmiRNA detected after 2 minutes at 40°, 50° or 60° C. Similar resultswere observed for miR23a, miR142, miR191, and miR451a. Let7a miRNA wasalso lost when samples were heated to 50° or 60° C. The loss of miRNA islikely due to RNase carry-over contamination with RIP, since blood isknown to contain extremely high levels of RNase.

Example 6. Release of miRNA Using Proteinase K Digestion

Proteinase K digestion was tested as a means to release miRNA afterAgo-RIP. RIP was performed with 0.2 ml plasma and 2.5 μg of biotinylatedanti-Ago2 antibody bound to 20 μl streptavidin magnetic beads. Post-RIP,beads were incubated in digestion buffer (20 μl of 10 mM Tris-HCL, pH8.0, 1.5 mM MgCl₂, 50 mM KCl, 10 mM DTT, 0.1% IGEPAL) containing 4 μlproteinase K (Sigma-Aldrich P4850) at room temperature or 37° C. for 10minutes with agitation, or at 65° C. for 2 minutes with agitation. Afterremoving the beads, Proteinase K was inactivated at 95° C. for 5 minutesand 5 μl of each proteinase K digest was added to a 10 μl polyA-tailingreaction for specific miRNA detection with Sigma-Aldrich's MystiCqRT-qPCR assays. For comparison, a parallel preparation of post-RIP beadswere extracted with QIAzol lysis reagent and purified with miRNeasySerum/Plasma (“total”, set at 100%). miRNA levels from RIP-proteinase Kwere expressed relative to those from RIP in which miRNAs were releasedusing the miRNeasy kit.

Release of miRNA using proteinase K digestion at room temperatureyielded more miRNAs than release at higher temperatures (FIG. 5). In allcases, a significant amount of total miRNA was lost. The loss was mostlikely due to residual RNAse in the sample.

A similar experiment was performed using pepsin digestion in buffers atpH 2, 3, or 4 for release of miRNA instead of proteinase K. miRNArelease using pepsin recovered less than 1% of miRNA (data not shown).

Example 7. Comparing RIP with Other Methods for miRNA Extraction fromBiofluids

miRNAs were isolated using biotinylated anti-Ago2 and streptavidinmagnetic beads and protease K release essentially as described inExample 6. For comparison, miRNAs was also purified directly from plasmausing the miRCury™ RNA Isolation Kit—Biofluids from Exiqon, or themiRneasy Serum/Plasma Kit from Qiagen (FIG. 6). These data show thatlet7a miRNA yields from RNA purified using Exiqon's kit were 3-4 timeshigher than that purified using Qiagen's kit. Yields of most miRNAsprepared using Ago-RIP and proteinase K release were intermediatebetween those of Exiqon and Qiagen in most experiments. Results forlet7a are shown in FIG. 6 and FIG. 7, but those for miR23a, miR142, andmiR191 were similar. On the other hand, yields of miR451a were similarfor Ago-RIP and Exiqon. miR451a requires Ago2 slicer activity forprocessing to a mature miRNA, and therefore, only occurs in Ago2complexes. Other miRNAs can associate with Ago1, Ago3, or Ago4 inaddition to Ago2. Since the antibody used is specific for Ago2, itisolates miR451a more efficiently than it does all other mature miRNAs.

Example 8. RIP with or Without Protease Inhibitors and RNAse Inhibitors

Ago-RIP was used to isolate miRNAs in the presence (+inh) or absence(−inh) of protease inhibitors and RNase inhibitors, and miRNAs werereleased from the beads with protease K. Ago-RIP performed in thepresence of inhibitors yielded more let7a miRNA than samples that werenot treated with inhibitors. Similar results were found for miR191.There was no significant difference for miR451a (FIG. 8).

Pretreatment of serum with IGEPAL with or without inhibitors was alsoperformed (+pre), and compared to addition of IGEPAL with or withoutinhibitors at the same time as addition of antibody beads (−pre). Theresults show that pretreatment of serum with protease and RNAseinhibitors did not improve yields of either let7a or miR451a whencompared to co-treatment (FIG. 8).

Example 9. miRNA Recovery with or Without Detergent

To determine the effect of detergent on the isolation of miRNAs, Ago-RIPwas performed (with 0.2 ml plasma) in the presence or absence of IGEPALdetergent using 10 μg of biotinylated anti-Ago2 antibody/streptavidinmagnetic beads. It was assumed that any miRNA isolated in the absence ofdetergent was free (i.e., not in a vesicle), and any isolated in thepresence of detergent was vesicular. Total miRNA was the level of amiRNA recovered from IGEPAL-treated plasma, and was set to 100%. FreemiRNA (miRNA not associated with vesicles) was the level of a miRNArecovered from plasma that was not treated with IGEPAL.Vesicle-associated miRNA was calculated as the level of a miRNA in thetotal miRNA sample subtracted by the level of said miRNA in the freemiRNA sample. FIG. 9 shows the free and vesicle-associated levels oflet7a, miR23a, miR142, and miR451a miRNAs. These results show thatdetergent treatment may be desirable to recover some miRNAs efficientlyfrom plasma by RIP.

Example 10. RIP is Scalable

RIP was performed with 0.2 ml plasma and 10 μg biotinylatedanti-Ago2/streptavidin beads, or 0.4 ml plasma and 20 μg biotinylatedanti-Ago2/streptavidin beads, followed by release with proteinase Kdigestion. For comparison, miRNAs were isolated from 0.2 ml of the sameplasma with Exiqon's miRCury RNA Isolation Kit—Biofluids. Total yieldsof let7a, miR191, and miR451a recovered with each preparation method areshown in FIG. 10. With Ago-RIP, twice as much plasma (i.e., 0.4 mlversus 0.2 ml) yielded 1.5-2-times as much of the miRNAs tested, whereascolumn-based kits (such as those from Exiqon and Qiagen) arecapacity-limited and recommend the use of no more than 0.2 ml of plasma.

Example 11. Minimum Incubation and Washing Times for RIP

RIP was performed with 0.2 ml plasma and 5 μg biotinylatedanti-Ago2/streptavidin beads, incubated with rotation at roomtemperature for 5, 15, 30, or 60 minutes. Those incubated for 5, 15, or30 min were all washed 5 times after the incubation was completed. Thosewith 60 min incubations were washed 5, 4, 3, 2, or 1 times with the RIPwash buffer. All were released with proteinase K. Yields for let7a arepresented in FIG. 11. The results show that an incubation period of morethan 15 minutes appears to be needed for maximum miRNA recovery underthe conditions used (e.g., type and amounts of antibody and beads), butonly one wash is needed before miRNA detection with Sigma-Aldrich'sMystiCq assays (polyA tailing, RT, qPCR). Similar results were obtainedfor miR122, miR191, and miR451a.

Example 12. RIP is Specific for miRNAs

The following example was performed to determine whether Ago-RIP isspecific for miRNAs or whether Ago-RIP also isolates other RNAs.Isolations were performed using Ago-RIP (S), Exiqon's miRCury RNAIsolation Kit—Biofliuds (E), or Qiagen's miRneasy Serum/Plasma Kit (Q)from 0.2 ml fresh plasma (experiments 1 and 2) or 0.2 ml frozen plasma(experiment 3). Experiment 1 was performed with 2.5 μg biotinylatedanti-ago2 antibody/20 μl streptavidin beads. Experiments 2 and 3 wereperformed with 2.5 μg biotinylated anti-ago2 antibody/20 μl streptavidinbeads. Proteinase K digestion essentially as described above in Example6 was used to release the miRNAs from the beads. Specific miRNAs (e.g.,let7a) and specific small nuclear or nucleolar RNAs (e.g., RNU6 orSNORD48) were detected using MystiCq RT-qPCR assays, and longer mRNAs orrRNAs (e.g., GAPDH, RN18S, RN28S) were detected using KiCqStart® RT-qPCR(Sigma-Aldrich) assays. Total RNA from HeLa cells (isolated using TRIReagent®BD) was used for quantitation standards.

As expected, miRNAs such as let7a were isolated using Ago-RIP or eitherof the column-based kits (see FIG. 12A). However, other small RNAs orlarge RNAs were not isolated by Ago-RIP but were isolated with Exiqonand Qiagen kits. As shown in FIG. 12B and FIG. 12C, little or no RNU6,SNORD48, GAPDH, RN18S, or RN28S RNAS were isolated using Ago-RIP, butall of these other types of RNA were isolated with the Exiqon and Qiagenkits. Thus, Ago-RIP specifically isolates only miRNAs.

Example 13. Use of Both Anti-Ago1 and Anti-Ago2 Antibodies Increases RIPYield

Since different miRNAs associate with different Ago proteins, it ispossible that yields of certain miRNAs could be improved through thecombined use of antibodies against different Ago proteins. Thus, miRNAswere isolated from 0.2 ml of plasma using 10 μg anti-Ago1 antibody/20 μlProtein A beads, 10 μg anti-Ago2 antibody/20 μl Protein A beads, or 20μl of a 1:1 mixture of each type of antibody bead. Release of the miRNAsfrom the beads was performed using QIAzol lysis reagent and purifiedwith the Qiagen kit essentially as described above in Example 3.Specific miRNAs (e.g., let7a, miR142-3p, miR122, miR191, and miR451a)were detected using MystiCq RT-qPCR assays.

As shown in FIG. 13, yields using both antibodies together wereapproximately the sum of each antibody used separately. For most miRNAs,the use of anti-Ago2 resulted in a greater yield than use of anti-Ago1.However, more miR122 was recovered when anti-Ago1 was used, which isconsistent with Turchinovich et al., 2012, RNA Biology 9(8):1066-75).Also, miR451a was only recovered with anti-Ago2, as explained above inExample 7.

What is claimed is:
 1. A method for isolating microRNA (miRNA), themethod comprising (a) contacting a biological sample with (i) at leastone surface active agent selected from an anionic surfactant, anon-ionic surfactant, and combinations of anionic surfactants and/ornon-ionic surfactants; (ii) an anti-Argonaut antibody, wherein thesurface active agent dissociates biological sample components and theanti-miRNA-binding protein reagent interacts with a miRNA-bindingprotein associated with miRNA to form immunoprecipitated miRNAcomplexes, and wherein the biological sample, the at least one surfaceactive agent, and the anti-Argonaut antibody are contacted at roomtemperature; and (b) contacting the immunoprecipitated miRNA complexes aproteinase K at room temperature to release miRNA from theimmunoprecipitated miRNA complexes without purification.
 2. The methodof claim 1, wherein the biological sample comprises vesicular miRNA andnon-vesicular miRNA.
 3. The method of claim 1, wherein the biologicalsample comprises a tissue sample.
 4. The method of claim 1, wherein theat least one surface active agent selected from polyoxyethylene (20)sorbitol monolaurate (TWEEN® 20); polyoxyethylene (20) sorbitolmonopalmitate (TWEEN® 40); polyoxyethylene (20) sorbitol monooleate(TWEEN® 80); sodium deoxycholate, sodium lauryl sulfate, andcombinations thereof.
 5. The method of claim 1, wherein the biologicalfluid sample is contacted with the at least one surface active agent andthe anti-Argonaut antibody simultaneously.
 6. The method of claim 1,wherein the biological sample, the at least one surface active agent,and the anti-Argonaut antibody are incubated for about 30 minutes. 7.The method of claim 1, wherein the anti-Argonaut antibody is attached toa solid support.
 8. The method of claim 7, wherein the solid support ismagnetic beads.
 9. The method of claim 1, wherein anti-Argonaut antibodyis capable of binding Ago1, Ago2, Ago3, and/or Ago4.
 10. The method ofclaim 1, wherein contact with the proteinase K proceeds for about 10minutes.
 11. The method of claim 1, wherein the miRNA released from theimmunoprecipitated miRNA complexes is free of other types of RNAmolecules.